The interaction mode of Di-n-octyl phthalate(DnOP) with human serum albumin(HSA) in physiological buffer(pH 7.4) was determined by multispectroscopic methods including fluorescence,Ultraviolet-visible(UV-vis) absorption,circular dichroism(CD) and Fourier transform infrared(FT-IR) spectroscopy,coupled with molecular modeling technique.Results obtained from the analysis of fluorescence titration indicated that the fluorescence quenching of DnOP for HSA was a static procedure resulting.The thermodynamic analysis of the binding data obtained at different temperatures indicated that the interaction between DnOP and HSA was mainly driven by hydrophobic interactions and hydrogen bonds,as the values of the entropy change(ΔS°) and the enthalpy change(ΔH°) were found to be 35.32 J?mol-1?K-1 and -9.13 kJ?mol-1,respectivelyThe site marker competitive experiments using different kinds of probes suggested a displacement reaction occurred between Eosin Y and DnOP with HSA.This result indicated that the binding site of DnOP to HSA located in the subdomain ⅡA(Site Ⅰ).Molecular modeling studies also proved that DnOP molecules inserted into the large hydrophobic activity of subdomain ⅡA by the hydrophobic interactions and hydrogen bond between oxygen atom of carboxide and residue His242.UV-vis absorption,CD and FT-IR spectra revealed that the binding of DnOP to HSA changed the second structure of HSA with a loss of α-helical content，which suggested a partial unfolding of the polypeptide chain of the protein in the presence of DnOP.